Open AccessMultifluorescence High‐Resolution Episcopic Microscopy for 3D Imaging of Adult Murine Organs
Author(s) -
Walsh Claire,
Holroyd Natalie A.,
Finnerty Eoin,
Ryan Sean G.,
Sweeney Paul W.,
Shipley Rebecca J.,
Walker-Samuel Simon
Publication year - 2021
Publication title -
advanced photonics research
Language(s) - English
Resource type - Journals
ISSN - 2699-9293
DOI - 10.1002/adpr.202100110
Subject(s) - optical sectioning , microscopy , resolution (logic) , biological specimen , light sheet fluorescence microscopy , biomedical engineering , fluorescence microscope , microscope , materials science , pathology , fluorescence , optics , computer science , artificial intelligence , scanning confocal electron microscopy , medicine , physics
3D microscopy of large biological samples (>0.5 cm 3 ) is transforming biological research. Many existing techniques require trade‐offs between image resolution, sample size, and method complexity. A simple robust instrument with the potential to conduct large‐volume 3D imaging currently exists in the form of the optical high‐resolution episcopic microscopy (HREM). However, the development of the instrument to date is limited to single‐fluorescent wavelength imaging with nonspecific eosin staining. Herein, developments to realize the potential of the HREM to become multifluorescent high‐resolution episcopic microscopy (MF‐HREM) are presented. MF‐HREM is a serial‐sectioning and block‐facing wide‐field fluorescence imaging technique, which does not require tissue clearing or optical sectioning. Multiple developments are detailed in sample preparation and image postprocessing to enable multiple specific stains in large samples and show how these enable segmentation and quantification of the data. The application of MF‐HREM is demonstrated in a variety of biological contexts: 3D imaging of whole tumor vascular networks and tumor cell invasion in xenograft tumors up to 7.5 mm 3 at resolutions of 2.75 μm, quantification of glomeruli volume in the adult mouse kidney, and quantification of vascular networks and white‐matter track orientation in adult mouse brain.